Focal adhesion kinase (FAK) is a non-receptor cytoplasmic tyrosine kinase that plays a key role in the regulation of proliferation and migration of normal and tumor cells. FAK associates with integrin receptors and recruits other molecules to the site of this interaction thus forming a signaling complex that transmits signals from the extracellular matrix to the cell cytoskeleton. Crk-associated substrate (CAS) family members appear to play a pivotal role in FAK regulation of cell migration. Cellular Src bound to FAK phosphorylates CAS proteins leading to the recruitment of a Crk family adaptor molecule and activation of a small GTPase and c-Jun N-terminal kinase (JNK) promoting membrane protrusion and cell migration. The relocalization of CAS and signaling through specific CAS family members appears to determine the outcome of this pathway. FAK also plays an important role in regulating cell cycle progression through transcriptional control of the cyclin D1 promoter by the Ets B and Kruppel-like factor 8 (KLF8) transcription factors. FAK regulation of cell cycle progression in tumor cells requires Erk activity, cyclin D1 transcription, and the cyclin-dependent kinase (cdk) inhibitor p27Kip1. The ability of FAK to integrate integrin and growth factor signals resulting in synergistic promotion of cell migration and proliferation, and its potential regulation by nuclear factor kappa B (NFkappaB) and p53 and a ubiquitously expressed inhibitory protein, suggest that it is remarkable in its capacity to integrate multiple extracellular and intracellular stimuli.

The highly invasive behavior of glioblastoma cells contributes to the morbidity and mortality associated with these tumors. The integrin-mediated adhesion and migration of glioblastoma cells on brain matrix proteins is enhanced by stimulation with growth factors, including platelet-derived growth factor (PDGF). As focal adhesion kinase (FAK), a nonreceptor cytoplasmic tyrosine kinase, has been shown to promote cell migration in various other cell types, we analysed its role in glioblastoma cell migration. Forced overexpression of FAK in serumstarved glioblastoma cells plated on recombinant (rec)-osteopontin resulted in a twofold enhancement of basal migration and a ninefold enhancement of PDGF-BBstimulated migration. Both expression of mutant FAK(397F) and the downregulation of FAK with small interfering (si) RNA inhibited basal and PDGF-stimulated migration. FAK overexpression and PDGF stimulation was found to increase the phosphorylation of the Crk-associated substrate (CAS) family member human enhancer of filamentation 1 (HEF1), but not p130CAS or Src-interacting protein (Sin)/Efs, although the levels of expression of these proteins was similar. Moreover downregulation of HEF1 with siRNA, but not p130CAS, inhibited basal and PDGF-stimulated migration. The phosphorylated HEF1 colocalized with vinculin and was associated almost exclusively with 0.1% Triton X-100 insoluble material, consistent with its signaling at focal adhesions. FAK overexpression promoted invasion through normal brain homogenate and siHEF1 inhibited this invasion. Results presented here suggest that HEF1 acts as a necessary and specific downstream effector of FAK in the invasive behavior of glioblastoma cells and may be an effective target for treatment of these tumors.

Focal adhesion kinase (FAK) is a non-receptor cytoplasmic-tyrosine kinase that is activated by several different cell surface receptors shown to be upregulated on glioblastoma cells (integrins alpha(v)beta3 and alpha(v)beta5, and the epidermal growth factor receptor). Activated FAK can signal through several different signaling pathways, which are reviewed here. Published data are summarized that have demonstrated 1) elevated FAK expression in anaplastic astrocytoma and glioblastoma tumor biopsy samples, 2) a role for FAK in the promotion of glioblastoma cell proliferation, survival and migration in vitro, and 3) a role for FAK in the promotion of glioblastoma cell proliferation in vivo in an animal model. The available data suggests that increased levels of FAK protein and activity may contribute to an increased ERK activity and cell proliferation in vivo in these tumors.

The objective of this study is to decipher the potentials of breast cancer cells that survive radiation exposure at clinical doses to acquire invasive and metastatic determinants. In this study we exposed estrogen receptor positive (MCF-7) and negative (MDAMB-231) breast cancer cells to clinical doses of low LET radiation including doses used as single fraction (2Gy), cumulative dose (10Gy) and scattered dose (10cGy). The cells were exposed to 137Cs source at a dose rate of 1.27 Gy/min. The mock irradiated cells were used as controls. The mRNA transcript level, protein expression and protein-protein interaction were analyzed by QPCR/RT-PCR, immunoblotting, immunoprecipitation and mammalian two-hybrid system, respectively. Intracellular nitric oxide (NO) levels were determined by immunofluorescence and electron spin resonance spectroscopy (ESR) using DAF-FM and Fe-MGD as NO traps, respectively. NO-dependent ER-α s-nitrosylation was determined through immunoprecipitating s-nitrosylated proteins from the cell lysates with an s-nitrosylated-cysteine IgG antibody followed by ER-α western blot analysis. Transactivation of ER-α was measured by EMSA and luciferase reporter assay. Cell invasion and migration were examined by co-culture system using thin inserts.Radiation induces eNOS expression and activation through the phosphorylation of eNOS at Ser1177 site resulting in increased bioavailability of NO in a dose- and time-dependent manner. The three-fold bioavailability of NO s-nitrosylates ER-α leading to a two-fold binding of ER-α to ERE. Paradoxically, this binding did not translate into the transcriptional activation of ER-α dependent gene regulation. Altered structure due to s-nitrosylation of ER-α contributes to enhanced cell invasion and cell migration. In consistent with these results, there was an associated 2.3-fold increase in the expression of MMP-2 and MMP-9 and corresponding TIMP-1 decreased expression leading to a 3.5-fold increase in MMP activity. Radiation, while controlling tumor growth, could simultaneously play a significant role in breast cancer relapse and metastasis through the activation of eNOS and the generation of NO. The bioavailability of NO s-nitrosylates ER-α leading to re-defining the genomic functions of ER-α and thereby impart metastatic transformation potentials to ER-α positive breast cancer cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6162.

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